1. Field of the Invention
The present invention relates to the composition of synthetic multivesicular lipid vesicles or liposomes encapsulating biologically active substances and to methods for their manufacture and use.
2. Description of Related Art
Because of the easily confused acronyms, multivcsicular liposomes (MVL) have been frequently confused with multilamellar vesicles (MLV), even among those familiar with the art. Nevertheless, the two entities are entirely distinct from each other. Multivesicular liposomes (MVL) are defined as liposomes containing multiple non-concentric chambers within each liposome particle, resembling a "foam-like" matrix; whereas multilamellar liposomes (also known as multilamellar vesicles or "MLV") contain multiple concentric chambers within each liposome particle, resembling the "layers of an onion". The distinctive structural features that clearly set MVL apart from MLV are easily seen by electron microscopy (FIG. 1).
In addition to the above two types of liposomes, unilamellar liposomes (also known as unilamellar vesicles or "ULV") that enclose a single internal aqueous compartment have been described. Unilamellar liposomes include small unilamellar vesicles (SUV) (Huang, Biochemistry 8:334-352, 1969) and large unilamellar vesicles (LUV) (Kim et al., Biochim. Biophys. Acta 646:1-10, 1981). Again, the distinctive structural features that clearly set MVL apart from unilamellar liposomes are easily seen by electron microscopy (FIG. 1).
The prior art describes a number of techniques for producing unilamellar and multilamellar liposomes; for example, U.S. Pat. No. 4,522,803 to Lenk; U.S. Pat. No. 4,310,506 to Baldeschwieler; U.S. Pat. No. 4,235,871 to Papahadjopoulos; U.S. Pat. No. 4,224,179 to Schneider; U.S. Pat. No. 4,078,052 to Papahadjopoulos; U.S. Pat. No. 4,394,372 to Taylor; U.S. Pat. No. 4,308,166 to Marchetti; U.S. Pat. No. 4,485,054 to Mezei; and U.S. Pat. No. 4,508,703 to Redziniak. A comprehensive review of various methods for preparation of unilamellar and multilamellar liposomes can be found in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:465-508, 1980. The prior art also describes methods for producing multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta 728,339-348,1983). In fact, the method of Kim, et al. (Biochim. Biophlys. Acta 728,339-348, 1983) is the only report that describes multivesicular liposomes, but the encapsulation efficiency of some of the small molecules such as cytarabine, also known as cytosine arabinoside or ara-C, was relatively low, and the release rate of encapsulated molecules in biological fluids at 37.degree. C. could not be modulated. The prior-art multivesicular liposomes result in in vivo release of the encapsulated biologically active substance over a period less than 24 hours following a single bolus injection into a mammal and could not be modulated, which severely limits the usefulness of the prior art.
The prior art by Crommelin et al. (Intl. J. Pharm. 16, 79-92, 1983) discloses that multilamellar liposomes encapsulating the biologically active substance doxorubicin should be prepared in the presence of a slightly acidic medium (pH 4-6.3), because such liposomes have a slow leakage rate of doxorubicin on storage. The present invention is not intended to teach about retention or leakage of the encapsulated biologically substances on storage of vesicles as taught by Crommelin et al. As will be apparent from the Description of Preferred Embodiments, the purpose of the present invention is to teach modulation of release of the biologically active substance over time relating to its site of application in the body, as anticipated by in vitro release testing in biologically-relevant media. Furthermore, the present invention teaches that the amount of hydrochloride added during the process of multivesicular liposome preparation will modulate the subsequent release rate of the biologically active substance from the liposome at the in vivo site of application. Additionally, the present invention differs from that disclosed by Crommelin et al. (supra) because Crommelin et al. use a distinct preparation process which results in liposomes having entirely different structures from those of the multivesicular liposomes. Specifically, Crommelin et al. make multilamellar liposomes containing multiple concentric chambers within each liposome particle; whereas the present art makes multivesicular liposomes containing multiple non-concentric chambers within each liposome particle.
Optimal treatment with many drugs requires maintenance of a drug level for a prolonged period of time. For example, optimal anti-cancer treatment with cell cycle-specific antimetabolites requires maintenance of a cytotoxic drug level for a prolonged period of time. Because it is a cell cycle phase-specific drug, cytarabine is a highly schedule-dependent anti-cancer drug. Because this drug kills cells only when they are making DNA, a prolonged exposure at therapeutic concentration of the drug is required for optimal cell kill. Unfortunately, the half-life of many drugs, including cytarabine, is very short after an intraperitoneal (IP), intravenous (IV), intrathecal (IT), intraarticular (IA), intramuscular (IM), or subcutaneous (SC) dose. To achieve optimal cancer cell kill with a cell cycle phase-specific drug like cytarabine, two major requirements need to be met: first, the cancer must be exposed to a high concentration of the drug without doing irreversible harm to the host; and second, the tumor must be exposed for a prolonged period of time so that all or most of the cancer cells have attempted to synthesize DNA in the presence of the drug.
Prior to the present invention, it has proven difficult, costly, and dangerous to provide prolonged concentration of cytarabine in the intrathecal space. The only way of achieving a prolonged cerebrospinal fluid (CSF) drug level in the case of cytarabine has been through continuous IT infusion using a drug pump. This method is not routinely employed because it carries a large risk of producing bacterial meningitis. The only way of achieving a prolonged plasma drug level in the case of cytarabine is through continuous intraveneous or sub-cutaneous infusion, both of which are inconvenient and costly. In searching for a long-acting preparation, investigators in the past have attempted to achieve this by chemical modification of the drug molecule to retard metabolism or covalent attachment of a hydrophobic moiety to retard solubilization. However, such manipulations have resulted in new toxic effects (Finkelstein, et al., Cancer Treat Rep 63:1331-1333, 1979), or unacceptable pharmacokinetic or formulation problems (Ho et. al., Cancer Res. 37:1640-1643, 1977). Therefore, a slow-release depot preparation which provides a prolonged and sustained exposure at a therapeutic concentration of a biologically-active substance is needed. The present invention is directed to the production process, composition, and use of such a preparation.